Evolution requires the generation of genetic diversity (diversity in nucleic acid) followed by the selection of those nucleic acids which encode beneficial characteristics. Because the activity of the nucleic acids and their encoded gene product are physically linked in biological organisms (the nucleic acids encoding the molecular blueprint of the cells in which they are confined), alterations in the genotype resulting in an adaptive change(s) of phenotype produce benefits for the organism resulting in increased survival and offspring. Multiple rounds of mutation and selection can thus result in the progressive enrichment of organisms (and the encoding genotype) with increasing adaptation to a given selection condition. Systems for rapid evolution of nucleic acids or proteins in vitro must mimic this process at the molecular level in that the nucleic acid and the activity of the encoded gene product must be linked and the activity of the gene product must be selectable.
In vitro selection technologies are a rapidly expanding field and often prove more powerful than rational design to obtain biopolymers with desired properties. In the past decade selection experiments, using e.g. phage display or SELEX technologies have yielded many novel polynucleotide and polypeptide ligands. Selection for catalysis has proved harder. Strategies have included binding of transition state analogues, covalent linkage to suicide inhibitors, proximity coupling and covalent product linkage. Although these approaches focus only on a particular part of the enzymatic cycle, there have been some successes. Ultimately however it would be desirable to select directly for catalytic turnover. Indeed, simple screening for catalytic turnover of fairly small mutant libraries has been rather more successful than the various selection approaches and has yielded some catalysts with greatly improved catalytic rates.
While polymerases are a prerequisite for technologies that define molecular biology, i.e. site-directed mutagenesis, cDNA cloning and in particular Sanger sequencing and PCR, they often suffer from serious shortcomings due to the fact that they are made to perform tasks for which nature has not optimized them. Few attempts appear to have been made to improve the properties of polymerases available from nature and to tailor them for specific applications by protein engineering. Technical advances have been largely peripheral, and include the use of polymerases from a wider range of organisms, buffer and additive systems as well as enzyme blends.
Attempts to improve the properties of polymerases have traditionally relied on protein engineering. For example, variants of Taq polymerase (for example, Stoffel fragment and Klentaq) have been generated by full or partial deletion of its 5′-3′ exonuclease domain and show improved thermostability and fidelity although at the cost of reduced processivity (Barnes 1992, Gene 112, 29-35, Lawyer et al., 1993, PCR Methods and Applications 2, 275). In addition, the availability of high-resolution structures for proteins has allowed the rational design of mutants with improved properties (for example, Taq mutants with improved properties of dideoxynucleotide incorporation for cycle sequencing, Li et al., 1999, Proc. Natl. Acad. Sci. USA 96, 9491). In vivo genetic approaches have also been used for protein design, for example by complementation of a polA strain to select for active polymerases from repertoires of mutant polymerases (Suzuki et al., 1996 Proc. Natl. Acad. Sci. USA 93, 9670). However, the genetic complementation approach is limited in the properties that can be selected for.
Recent advances in molecular biology have allowed some molecules to be co-selected in vitro according to their properties along with the nucleic acids that encode them. The selected nucleic acids can subsequently be cloned for further analysis or use, or subjected to additional rounds of mutation and selection. Common to these methods is the establishment of large libraries of nucleic acids. Molecules having the desired characteristics (activity) can be isolated through selection regimes that select for the desired activity of the encoded gene product, such as a desired biochemical or biological activity, for example binding activity.
WO99/02671 describes a method for isolating one or more genetic elements encoding a gene product having a desired activity. Genetic elements are first compartmentalised into microcapsules, and then transcribed and/or translated to produce their respective gene products (RNA or protein) within the microcapsules. Alternatively, the genetic elements are contained within a host cell in which transcription and/or translation (expression) of the gene product takes place and the host cells are first compartmentalised into microcapsules. Genetic elements which produce gene product having desired activity are subsequently sorted. The method described in WO99/02671 relies on the gene product catalytically modifying the microcapsule or the genetic element (or both), so that enrichment of the modified entity or entities enables selection of the desired activity.